Annexin A4 is involved in proliferation, chemo-resistance and migration and invasion in ovarian clear cell adenocarcinoma cells

Abstract

Ovarian clear cell adenocarcinoma (CCC) is the second most common subtype of ovarian cancer after high-grade serous adenocarcinomas. CCC tends to develop resistance to the standard platinum-based chemotherapy, and has a poor prognosis when diagnosed in advanced stages. The ANXA4 gene, along with its product, a Ca(++)-binding annexin A4 (ANXA4) protein, has been identified as the CCC signature gene. We reported two subtypes of ANXA4 with different isoelectric points (IEPs) that are upregulated in CCC cell lines. Although several in vitro investigations have shown ANXA4 to be involved in cancer cell proliferation, chemoresistance, and migration, these studies were generally based on its overexpression in cells other than CCC. To elucidate the function of the ANXA4 in CCC cells, we established CCC cell lines whose ANXA4 expressions are stably knocked down. Two parental cells were used: OVTOKO contains almost exclusively an acidic subtype of ANXA4, and OVISE contains predominantly a basic subtype but also a detectable acidic subtype. ANXA4 knockdown (KO) resulted in significant growth retardation and greater sensitivity to carboplatin in OVTOKO cells. ANXA4-KO caused significant loss of migration and invasion capability in OVISE cells, but this effect was not seen in OVTOKO cells. We failed to find the cause of the different IEPs of ANXA4, but confirmed that the two subtypes are found in clinical CCC samples in ratios that vary by patient. Further investigation to clarify the mechanism that produces the subtypes is needed to clarify the function of ANXA4 in CCC, and might allow stratification and improved treatment strategies for patients with CCC.

Figure 1. ANXA4 expression visualized in surgically removed ovarian tumors using IHC.

(A) Representative images for ANXA4 immunohistochemical (IHC) scores are shown. Each scale bar represents 200 µm. (B) IHC scores were significantly high in clear cell carcinoma compared with tumors with low malignant potential (LMP) and carcinomas (P<0.05). ca, carcinoma; diff, differentiated.

Figure 2. Establishment of ANXA4 knockdown clones and the effect on cell proliferation.

(A) Western blotting for ANXA4, with β-actin as a loading control. Negative control clones, expressing non-ANXA4 mRNA-targeting shRNA are designated as NC [clone number] with data for parental cells. (B, C) Proliferation was examined by a WST-1 assay as described in the text. Experiments for clones and parental cells were performed in triplicate.

Figure 3. Effect of ANXA4 knockdown on carboplatin and paclitaxel sensitivity.

Sensitivity of OVTOKO cell to carboplatin (A) and paclitaxel (C); and of OVISE cells to carboplatin (B) and paclitaxel (D). Drug concentrations are indicated below each panel; cell viability under each concentration was evaluated by a WST-1 assay as described in the text. Experiments for clones and parental cells were performed in triplicate.

Figure 4. Effect of ANXA4 knockdown on cell migration and invasion and western blotting for membrane proteins RHAMM and LAMP2.

OVISE cell migration (A) and invasion (B); and OVTOKO cell migration (C) and invasion (D). Experiments for clones and parental cells were performed in triplicate. (E) Expression of membrane proteins RHAMM and LAMP2 was demonstrated by western blotting in OVISE and OVTOKO cells. β-actin was used as a loading control in western blotting.

Figure 5. Demonstration of the two ANXA4 subtypes with different IEPs in surgically removed CCC samples.

(A) Table of clinical CCC samples examined by 2D-PAGE and bar graph (on right) of their ratios of ANXA4 subtypes. pT, pathological T stage; Ope, surgical removal of the tumors; +, post-operational chemotherapy; TC, combined paclitaxel and carboplatin chemotherapy; DC, combined docetaxel and carboplatin chemotherapy; NAC, neoadjuvant chemotherapy. (B) Representative images of 2D-PAGE followed by western blotting with a IHC image of the corresponding case (scale bar: 200 µm).

Figure 6. Involvement of phosphorylation, Ca²⁺ conjugation, and acetylation in production of two ANXA4 subtypes with different IEPs.

(A) OVISE cells containing both acidic and basic ANXA4 subtypes were used to study effects of deacetylation, Ca²⁺ chelation, and dephosphorylation on subtype ratios. Each 2D-PAGE and western blot image for ANXA4 shows the treatment (above the panel), and the signal intensity (below each spot), as evaluated by an image analyzer ImageQuant LAS 4000 (GE Healthcare Life Science, UK). The left side spot indicates the acidic form and the right side spot indicates the basic form of ANXA4 respectively. (B) Acidic/basic subtype ratios were derived from signal intensities, and are shown in a bar of 100%.